Method for manufacture of lactate biosensing strip

ABSTRACT

We provide a lactate biosensing strip comprising a working electrode and a reference electrode, the two electrodes being deposited on an electrically insulated base support, the working electrode being formed by immobilizing lactate oxidase and an electron mediator on an inorganic graphite matrix and the graphite layer being deposited on a silver layer of the working electrode and the reference electrode being formed by depositing silver chloride on a silver layer of the reference electrode.

RELATED APPLICATION

This is a division of our copending commonly assigned application10/342,303 filed Jan. 15, 2003 now allowed.

FIELD OF THE INVENTION

The invention relates to a lactate biosensing strip for the measurementof lactate solution. The present invention also relates to a method forthe manufacture of a novel lactate biosensing strip and to the usethereof for lactate sensing.

BACKGROUND OF THE INVENTION

Physicians rely on personal examination and clinical laboratory resultsto determine the presence and concentration of biological analytes incritical care patients. Clinical laboratories offer a wide range ofautomated systems for high-volume testing and analytical support in awell controlled, high quality environment. However, clinicallaboratories can not provide the immediate results needed to properlytreat trauma and multi organ dysfunction/failure patients.

To meet the clinical need for immediate test results, severaltechnologies are emerging for testing using reliable, automatedanalyzers at the patient's bedside including electrochemical biosensors,optical fluorescence sensors, paramagnetic particles for coagulationtest systems, and micromachined devices for both chemical andimmunochemical testing. These technologies have allowed multi-analytechemistry panels to be performed rapidly and have addressed previousobstacles such as calibration of test devices.

These tests can be classified as: 1) in vitro, which is performed at thebedside; 2) ex vivo or para vivo, which is performed at wrist-side; and3) in vivo, which is performed inside the patient. Such tests offerindirect cost efficiencies and savings such as reduced labor costs,decreased blood identification and transport errors, and reduced patientcomplications.

In vitro or bedside devices are used typically in several departments ofthe hospital including intensive care units; operating rooms; emergencydepartments (ER); interventional departments; general patient caredepartments; and outpatient surgery and ambulatory care units. In vitrodiagnostic tests offer a wide range of diagnostic tests, similar to theclinical laboratory. In vitro diagnostic test systems typically are notconnected on-line to the patient and require an operator for bloodsampling.

Key categories of diagnostic test in the diagnostic market includearterial blood gases, blood chemistries, blood glucose, coagulation,drugs-of-abuse testing, hemoglobin, hematocrit, infectious diseases, andtherapeutic drug monitoring. Other categories include cancer markers,cardiac markers, cholesterol detection, immunodiagnostics, infectiousdisease detection, lactate, and thrombolytic monitoring.

Ex vivo diagnostics use external sensors for on-line real-time testingwith little to no blood loss. Typically, sampled blood flows through aclosed system to minimize blood contact. Ex vivo systems minimizeproblems associated with in vivo sensors, including clotting,inaccuracy, calibration drift, and an inability to recalibrate once inthe patient. U.S. Pat. No. 5,505,828 discloses an exemplary ex vivosystem.

In vivo diagnostics offer considerable potential in the treatment ofmost critical and unstable patients. Although many companies aredeveloping in vivo sensors, technical hurdles have thus far kept in vivosensors from common commercial use.

Ex vivo and in vivo diagnostics, since they are on-line systems, canreduce quality control and information integration errors that occurwith clinical or in vitro tests. Quality control errors are commonly dueto operator errors, not instrument errors or device failures. Exemplaryerrors include inappropriate specimen volume, inaccurate calibration,use of deteriorated test strips, inadequate validation, insufficientinstrument maintenance, bad timing of the test procedure, and use of thewrong materials. Clinical information system integration allows testdata collected at the bedside to be put directly into the patientrecord. This improves the efficiency of the patient management process,allowing the integration of the laboratory's information system andclinical information systems, providing a “seamless” flow of all typesof patient information.

Lactate is the byproduct of carbohydrate metabolism and product ofglycolysis (pyrovate) is converted into lactate under an aerobiccondition i.e. deficiency of oxygen in cells. Lactate estimations aretherefore important in respiratory disorder, heart ailment, labordiseases etc. normal concentration of lactate in human blood is in therange of 1.2 to 2.7 mM.

Procedure for lactate determination for example, has employed a varietyof chemical and physical technique. Traditional assay involves chemicaltreatment of lactate in human blood and thereby converting it into colorproducts which can be measured spectrophotometrically, the methodsconsists in reacting the blood under test with enzyme namely lactatedehydrogenise (LDH). In such process absorbance at 340 nm is measureddue to the NADH formation, it becomes a measurement of lactateoriginally present in blood.

U.S. Pat. No. 6,117,290 discloses an on-line lactate sensor arrangement.The sensor arrangement includes a lactate sensor, a catheter forwithdrawing a test sample, and a first fluid flow line provided fluidcommunication between the lactate sensor and the catheter. The sensorarrangement also includes a source of sensor calibration andanticoagulant solution, and second fluid flow line providing fluidcommunication between the source of sensor calibration and anticoagulantsolution and the lactate sensor.

In practice there are some difficulties in adopting such a detectionprocedure for use with blood sample. The disadvantage of such methods,include, lack of specificity, difficulty of standardization requirementof large amount of blood and use of unstable and corrosive regents. Suchmethods also involve optical detection and are therefore expensive andtime consuming. Additionally, the samples must be prepared. Anotherdisadvantage is that the measurement of lactate level by prior artmethods need to be done in laboratory by qualified personnel.

Asha Chaubey et al disclose in Electrochimica Acta. Vol 46, 723-729(2000) the immobilization of lactate dehydrogenase on electrochemicallyprepared polypyrrole polyvinyl sulphonate composite films. The responsetime reported is about 40 seconds and a shelf life of about 2 weeksunder refrigerated conditions. In another disclosure (Asha Chaubey et alAnalyticla Chimica Acta Vol 49, 98-103, 2000), the immobilization oflactate dehydrogenase on conducting polyaniline films is disclosed. Thelinearity of response is shown from 0.1 mM to 1 mM lactate concentrationwith a shelf life of about 3 weeks under refrigerated conditions. It ispreferable to obtain sensors with longer shelf life and shorter responsetime.

Accordingly, it is important to provide a lactate biosensing strip thatcan overcome the disadvantages of the prior art without losing out onefficiency and accuracy of measurement.

OBJECTS OF THE INVENTION

The main object of this invention is to provide a novel lactatebiosensing strip for the measurement of lactate in aqueous medium.

It is another object of the invention to provide a lactate biosensingstrip which performs rapidly and accurately the estimation of lactate inan aqueous medium.

It is yet another object of the invention to provide a lactatebiosensing strip which is low cost and is capable of being used by evennon-medical persons.

A further object of this invention is an assay, which can be performedwithout the need for elaborate preliminary treatment of blood sample.

Another object of this invention is to provide a lactate biosensingstrip, which has a high activity of 75%.

Still another object of this invention is to provide a lactate-sensingstrip, which is capable for providing a reading at site.

SUMMARY OF THE INVENTION

Lactate biosensing strips have many advantages over traditional methods,such as fast response, small size convenience, specificity of response,lack of need of any sample preparation, low cost and high sensitivity ofmeasurement. The main advantage of this sensor over the traditionalmethod is sample operation it can be done by ordinary person.

The present invention provides a lactate biosensing strip for use in theassay of lactate in a sample, said sensor comprising a dry strip sensorof an electrically conducting material having at least:

-   i. an external surface.-   ii. a screen printed reference electrode and-   iii. a screen-printed working electrode.

Accordingly, the present invention provides a lactate biosensing stripcomprising a working electrode and a reference electrode, the said twoelectrodes being deposited on an electrically insulated base supportwherein the working electrode being formed by immobilizing an enzymelactate oxidase and an electro mediator on an inorganic graphite matrixand the said graphite layer being deposited on a silver layer and thereference electrode being formed by depositing silver chloride on ananother silver layer.

In an another embodiment the biosensing strip further comprises

-   i. an electrically insulated base support (1),-   ii. a pair of first and second silver layers deposited thereon (2)    separated by an appropriate space between the two said layers,-   iii. a pair of graphite layers, each one of said pair of graphite    layers being deposited on one respective silver layer and being    electrically connected to said respective silver layer (2),-   iv. the first silver layer being covered fully by the respective    graphite layer,-   v. the second silver layer being covered partly in the middle    thereof with the respective graphite layer after leaving the    connecting terminal and working zone area uncovered,-   vi. the uncovered working zone of said second silver electrode layer    being deposited with silver chloride (4),-   vii. lactate oxidase being deposited with a mediator on the working    zone of graphite layer covering the first silver layer (5),-   viii. the said silver/silver chloride layer forming reference    electrode (4) and enzyme with mediator layer forming working    electrode (5) being supported on said support (1),-   ix. the working zone of reference electrode (4) and working    electrode (5) being covered with a hydrophilic membrane.

In one embodiment of the invention, the electrically insulated basesupport used is made of polyvinyl chloride.

In one embodiment of the invention the distance between the silverlayers is in the range of 0.5 to 1 mm

In another embodiment of the invention, the thickness of each silverlayer is in the range of 15 to 25 microns.

In another embodiment of the invention the electron mediator layercomprises a layer of potassium ferricyanide or ferrocene.

In another embodiment of the invention, the hydrophilic membrane is madeof nylon or polyester.

In another embodiment of the invention, the working zone are ofelectrode is a target area used for dispensing the analyte sample

In another embodiment of the invention the connecting terminal zone areaof electrode is an area used for the connectivity of electrode to anelectrometer

The lactate biosensing strip of the invention shows an activity of 75%and a response time for lactate detection is in the range of 30 to 40seconds. The shelf life of the strip of the invention is about 4 monthsunder refrigerated conditions. Under ambient conditions (25 to 30° C.)the shelf life of the biosensing strip is seen to about 2 months. Thestrip of the invention is disposable.

The invention also relates to a method for the manufacture of a lactatebiosensing strip said strip comprising an electrically insulated basesupport (1), a pair of isolated first and second silver layers depositedthereon (2), a pair of graphite layers, each one of said pair ofgraphite layers being deposited on one respective silver layer andelectrically connected to said respective silver layer (2), the firstsilver layer being covered fully by the respective graphite layer, thesecond silver layer being covered partly in the middle thereof with therespective graphite layer leaving the connecting and working zone areaof the said layer uncovered, a Ag/AgCl electrode (4) provided on top ofthe working area of said second silver electrode layer, lactate oxidasedeposited with a mediator on the working area of graphite layer coveringthe first silver layer (5), the said silver/silver chloride electrode(4) and enzyme with mediator working electrode being supported on saidsupport (1), said process comprising

-   (a) depositing a pair of silver layers on an electrically insulated    base support by any conventional method;-   (b) depositing a pair of graphite layers on said silver layers by    any conventional method, each of said silver layers being deposited    with one graphite layer, a first graphite layer completely covering    the first silver layer, and the second graphite layer covering the    second silver layer only in part of the surface thereof that is away    from the surface facing the base support;-   (c) depositing a silver chloride layer on the second silver layer on    the part thereof that is not deposited with a graphite layer to    obtain a silver/silver chloride electrode;-   (d) adsorbing physically lactate oxidase enzyme with an electron    mediator on the first graphite deposited silver layer to obtain a    working electrode; and-   (e) applying an outer hydrophilic membrane on the above said first    reference electrode and second working electrode to obtain the    desired pair of electrodes on an electrically insulated base support    in a single assembly.

In one embodiment of the invention the electrically insulated basesupport comprises of polyvinyl chloride.

In another embodiment of the invention said silver layer used is appliedby the step of screen-printing.

In another embodiment of the invention, said graphite layer used isapplied by the step of screen-printing.

In another embodiment of the invention, the sample being tested is anaqueous lactate solution or blood sample in an amount of 25 to 30 μL.

In another embodiment of the invention, the electron mediator used isselected from potassium ferricyanide and ferrocene.

In another embodiment of the invention, connecting terminal zone area ofelectrode is an area used for the connectivity of electrode to anelectrometer

In another embodiment of the invention, the hydrophilic membrane is madeof nylon or polyester.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a schematic representation of the biosensing strip of theinvention.

FIG. 2 is the response curve of the lactate biosensing strip of theinvention for standard lactate test samples.

FIG. 3 shows the calibration curve for the sensor against standardlactate test samples prepared in a laboratory.

FIG. 4 shows the shelf life stability characteristics of the lactatestrip of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As shown in FIG. 1, the invention comprises an electrically insulatedbase support (1) for supporting an electrode assembly (2), (3), (4) and(5). The electrode assembly comprises two electrode systems, a workingelectrode system (2), (3) and (5) consisting of a silver layer with agraphite layer deposited thereon and an enzyme and mediator layeradsorbed in the inorganic matrix. The other electrode assembly comprisesa reference electrode comprising a silver layer partly deposited with agraphite layer and a silver/silver chloride layer thereon FIG. 1 showsthe PVC sheet (i) which comprises the supporting substrate for theelectrode. Conducting silver tracking (ii) is the screen-printedconducting graphite layer onto the surface of conducting silver tracking(iii) for the connection of the sensor to read out apparatus. The targetarea consists of the working electrode (iv) and the reference electrode(v) applies to the end of tracking by screen-printing. An insulatedlayer is applied over the printed electrode to give them protection; themass can be coated with one or more legends. The conducting graphitetrack (ii) does not extend to the complete length of the silver trackand the reference electrode.

To achieve calibration of the biosensing strip, the strip was used todetect currents when the lactate solutions were used in concentrationsof 1 to 8 mM. The current measured for each of the concentrations wasmeasured and plotted in FIG. 2. In FIG. 2, curve (1) is the responsecurve for 1 mM lactate solution, curve (2) is for 2 mM solution, curve(3) is for 4 mM solution, curve (4) is for 6 mM solution and curve (5)is for 8 mM solution. This shows that the biosensing strip of theinvention can be used to measure lactate in a blood sample if the rangelies in the region of 1 to 8 mM in a subject. The sensitivity of thesystem in terms of the response time to attain a stable current valuewas determined by analyzing the strip time variation of current. Thiscomprised initiating current measurement from the time of putting thedrop of standard test solution on the strip to the time when the currentasymptotically reaches a stable value. It was observed (FIG. 3) that thecurrent attains the stable value in 30 to 40 seconds.

Shelf life characteristics were determined by measuring the current dueto a known lactate concentration on strips stored for different periodsof time. The data is given in FIG. 4. In FIG. 4, curve (1) is for stripsstored under refrigerated conditions (at 4° C.) while curve (2) is forstrips stored at 25-30° C.

The invention also provides a process for producing a lactate sensorstrip which comprise in forming a first and second electrode on asubstrate by applying a layer of silver for each of said electrodes insaid electrode, applying a layer graphite on the handling zone of saidsecond electrode to silver chloride, applying a mediator and enzyme onthe graphite layer of the working zone of the first electrode. An outerhydrophilic membrane is applied zone of said first electrode. The silverlayers and the graphite layers are preferably applied by the step ofscreen-printing.

The main feature of this invention is that the sensor is a dry stripsensor. It is found that a similar mix of reagents employed in a wetsensor system did not give good result across a desired range ofdetectable lactate concentration.

This invention comprises a substrate for supporting an electrodeassembly said electrode assembly comprising two electrode systems, oneworking electrode and another one as a reference electrode supported onsaid substrate and disposed in a spaced relationship to each other. Thelactate sensing strip comprising of a substrate for supporting a firstor working electrode and second or reference electrode, said electrodedisposed in a spaced relationship to each other.

The first electrode is a working electrode and has a terminal extendingin to a working zone through a handling zone. The second electrode is areference electrode and has a terminal extending in to a working zonethrough a handling zone. In both cases, the respective terminals are ofa material different to the base conducting layer of said first andsecond electrodes.

Commercially obtained lactate oxidase is mixed in a phosphate buffer,then proper amount of this solution is injected onto a preprintedworking electrode. This solution is allowed to dry in allow temperature,followed by

-   i. printing of conducting tracking-   ii. printing of reference electrode-   iii. printing of working electrode-   iv. fixing of membrane onto electrode.

The working and reference electrode each comprise a base conductinglayer of silver material along the handling and working zone. A graphitelayer is deposited on the silver layer of the working electrode andextends to the terminal; the graphite layer is applied on the handlingzone of the reference electrode and extends to the terminal. Ag/AgCl isdeposited on the target area of the reference electrode. Workingelectrode comprising conducting surface carrying mediator compound andlactate oxidase enzyme. Mediator compound transfer electrodes from theenzyme to the electrode, when such catalytic activity takes place. Ahydrophilic membrane must be provided on the working zone of saidelectrode. It appears that the surfactant serves to break up thelipoprotein complex of blood and lactate is then oxidized to thepyruvate by the lactate oxidase. The mediator compound iselectrochemically reduced at the electrode producing a currentmeasurable at the electrode, which current is relative to the activityof the lactate oxidize and hence the amount of lactate present in thesample this current is generated through a serious of coupled reactionsL-Lactate+LOD_((ox)) - - - Pyruvate+LOD_((red))LOD_((red))+Me_((ox)) - - - LOD_((ox))+Me_((re))

The redox mediator is oxidized at the base electrode and the currentproportional to the lactate concentration. The current can be measuredby any conventional electronic system.

The following examples are given by the way of illustration andtherefore should not constitute to limit the scope of the presentinvention.

EXAMPLE 1 Preparation of Graphite Paste with Mediator

100 mg of graphite powder and polyvinyl pyrrolidon (binder) was mixedwith 0.01M Potassium ferricyanide (mediator) in ethylene glycolmonobutyl ether to prepare screen printable working electrode graphitepaste.

EXAMPLE 2 Preparation of Dry Strip

Commercially obtained lactate oxidase solution (2 μL) containing 2 U oflactate oxidase was physically adsorbed on the mediator mixed graphiteelectrode strip and was kept over night to dry at 25° C. The dry stripelectrode was covered with a hydrophilic nylon membrane. Before themembrane was applied, it was placed in 10% surfactant (Tween 80)solution in distilled water for some time the dried membrane was thenfixed over the strip.

EXAMPLE 3 Preparation of Lactate Standard Lactate Solutions

Stock lactate solution 10 mM was prepared in 0.1M phosphate buffer.Standard solutions of 2 mM, 4 mM, 6 mM and 8 mM were prepared bydiluting the stock solution with phosphate buffer.

EXAMPLE 4 Preparation of Enzyme Stock Solution

15 mg of enzyme lactate oxidase was dissolved in 100 μl of 0.1Mphosphate buffer to get the concentration 5 U/μl to get the workingenzyme solution, the stock solution was further diluted to 1 U/μl.

EXAMPLE 5 Immobilization of Enzyme on the Mediator Mixed Graphite DryStrip

2 μl of enzyme solution containing 2 U of lactate oxidase was physicallyadsorbed on the mediator mixed graphite electrode strip and was keptover night to dry at 25° C. The said dry strip electrode was covered bya hydrophilic nylon membrane. Before applying the membrane, it wasplaced in 10% surfactant (Tween 80) solution in distilled water for sometime and then dried membrane was fixed over the strip.

EXAMPLE 6 Enzyme Activity

Sigma protocol for activity of lactate oxidase was used to estimate thelactate oxidase activity. The basic principle is that lactate oxidaseconverts l-lactate to pyruvate and H₂O₂. H₂O₂ is subsequently convertedinto a colored dye by peroxidase in the presence of 4-amino antipyrine(4AAP) and dimethylaniline(DMA).

In the optimum conditions of temperature=37° C. and pH=6.5, the dyeabsorbs at 565 nm at the light path of 1 cm.

The activity of the immobilized enzyme was calculated according to thefollowing formula:U cm⁻² =AV/εt sWhere A is the change in absorbance before and after incubation

V is the total volume (3 ml)

ε is the milimolar extinction coefficient of Quinonediimine dye at 565nm (35.33)

t is the reaction time (10 min)

s is the surface area of the enzyme electrode

The enzyme activity of immobilized LOD on the working graphite strip wasfound to be 75%.

EXAMPLE 7 Amperometric Response Studies

The lactate biosensing strip comprising enzyme(LOD) immobilized ongraphite as working electrode and Ag/AgCl reference electrode isconnected to the input of the electrometer was polarized at a biasvoltage of 0.4V for the measurement of amperometric calibration responseto lactate (1-8 mM)(FIG. 2). A maximum current of 60 μA was obtained for8 mM lactate solution above which no significant change in current couldbe observed. The response time for lactate solution (1-8 mM) was foundto be 40 seconds for each concentration of lactate (FIG. 3). Resultswere found to be reproducible to within 5%. Following principle wasinvolved in the amperometric measurements.

ADVANTAGES OF THE INVENTION

1. The lactate biosensing strip provides a quick estimation of lactatein a sample

2. the shelf life of the sample is 4 months under refrigeratedconditions.

3. the strip has a linear response in a lactate concentration of 1 to 8mM.

4. the strip is disposable without causing any environmental hazard.

5. the strip is easily used even by people without any formal medicaltraining.

1. A method for preparing a lactate biosensing strip comprising anelectrically insulated base support (1), a pair of isolated first andsecond silver layers deposited thereon (2), a pair of graphite layers,each one of said pair of graphite layers being deposited on onerespective silver layer and electrically connected to said respectivesilver layer (2), the first silver layer being covered fully by therespective graphite layer, the second silver layer being covered partlyin the middle thereof with the respective graphite layer after leavingthe terminal and working zone uncovered, a Ag/AgCl electrode (4)provided on top of the working zone of said second silver electrodelayer, lactate oxidase deposited with an electron mediator on theworking zone of graphite layer covering the first silver layer (5), thesilver/silver chloride electrode (4) and enzyme with mediator layerworking electrode (5) being supported on said support (1), the workingzone of silver/silver chloride reference electrode (4) and workingelectrode (5) being covered with a hydrophilic membrane, said lactatebiosensing strip being disposable, said method comprising: (a)depositing a pair of silver layers on an electrically insulated basesupport; (b) depositing two graphite layers on each of said two silverlayers, each of said two silver layers being deposited with one graphitelayer, a first graphite layer completely covering the first silverlayer, and the second graphite layer covering the second silver layeronly in part of the surface thereof that is away from the surface facingthe base support; (c) depositing a silver chloride layer on the secondsilver layer on the part thereof that is not deposited with a graphitelayer to obtain a silver/silver chloride electrode; (d) adsorbingphysically lactate oxidase enzyme with an electron mediator on the firstgraphite deposited silver layer to obtain a working electrode; and (e)applying an outer hydrophilic membrane on said first reference electrodeand second working electrode to obtain a pair of electrodes on anelectrically insulated base support in a single assembly.
 2. The methodas claimed in claim 1, wherein the electrically insulated base supportis polyvinyl chloride.
 3. The method as claimed in claim 1, wherein thesilver layer is applied by screen-printing.
 4. The method as claimed inclaim 1, wherein the graphite layer is applied by screen-printing. 5.The method as claimed in claim 1, further comprising testing a samplewhich is an aqueous lactate solution or blood sample in an amount of 25to 30 μL.
 6. The method as claimed in claim 1, wherein the electronmediator is potassium ferricyanide or ferrocene.
 7. The method asclaimed in claim 1, wherein a working zone of an electrode is a targetarea used for dispensing an analyte sample.
 8. The method as claimed inclaim 1, wherein a connecting terminal of the reference electrode is anarea used for connecting the reference electrode to an electrometer. 9.The method as claimed in claim 1, wherein the hydrophilic membrane ismade of nylon or polyester.
 10. A method for preparing a two electrodebiosensing strip for measuring lactate, said method comprising: (a)depositing a pair of silver layers on an electrically insulated basesupport; (b) depositing a pair of graphite layers on said silver layers,each of said silver layers being deposited with one graphite layer, afirst graphite layer completely covering the first silver layer, and asecond graphite layer covering only a part of the second silver layerthat is away from the surface facing the base support; (c) depositing asilver chloride layer on a part of the second silver layer that is notdeposited with a graphite layer to obtain a reference electrode; (d)adsorbing physically lactate oxidase with an electron mediator on thefirst graphite layer to obtain a working electrode; and (e) coveringsaid reference electrode and said working electrode with a hydrophilicmembrane to obtain said two-electrode biosensing strip in a singleassembly.
 11. The method as claimed in claim 10, wherein theelectrically insulated base support is polyvinyl chloride.
 12. Themethod as claimed in claim 10, wherein each silver layer is applied byscreen-printing.
 13. The method as claimed in claim 10, wherein eachgraphite layer is applied by screen-printing.
 14. The method as claimedin claim 10, further comprising testing a sample which is an aqueouslactate solution or blood sample in an amount of 25 to 30 μL.
 15. Themethod as claimed in claim 10, wherein the electron mediator ispotassium ferricyanide or ferrocene.
 16. The method as claimed in claim10, wherein a working zone of an electrode is a target area used fordispensing an analyte sample.
 17. The method as claimed in claim 10,wherein a connecting terminal of the reference electrode is an area usedfor connecting the reference electrode to an electrometer.
 18. Themethod as claimed in claim 10, wherein the hydrophilic membrane is madeof nylon or polyester.